ANALYSIS OF ALLERGENS AND USEFUL GENES IN BUCKWHEAT SEED STORAGE PROTEINS

• Project/Area Number: 15580010
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Crop science/Weed science
• Research Institution: HOKKAIDO UNIVERSITY
• Principal Investigator: FUJINO Kaien Hokkaido Univ., Grad.School of Agr., Lecturer, 大学院・農学研究科, 講師 (80229020)
• Co-Investigator(Kenkyū-buntansha): MASUDA Kiyoshi Hokkaido Univ., Grad.School of Agr., Prof, 大学院・農学研究科, 教授 (60157203)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,800,000 (Direct Cost: ¥3,800,000); Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000); Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
• Keywords: buckwheat / rutin / allegen / seed storage protein / tatary buckwheat / glucosidase / rutinase / アレルギー

Research Abstract

Buckwheat (Fagopyrum esculentum M.) gene EL0847 that showed an increase in transcript during the early stages of seed development was isolated from immature seeds harvested 14 days after pollination by differential screening. The deduced amino acid sequence of EL0847 shows several repeats of alanine, glutamate and valine which suggests a structural role for the encoded protein, and reveals similarity to the deduced amino acid sequence of Hev b5, which is believed to be a major allergen from natural rubber latex. In an E.coli. expression system, the translation product of a cDNA encoding the EL0847 was not recognized by sera from allergic patients.
Based on the N-terminal amino acid sequence of the rutinase which showed flavonol 3-glucosidase activity, a degenerate primer were synthesized and 3' RACE PCR amplification was carried out. The deduced amino acid sequence of rutinase gene shows similarity to the beta-glucosidase in plants. Antiserum was raised against the translation product of a cDNA encoding the rutinase in an E.coli. expression system and immunoblotting of total protein from tatary buckwheat seeds (Fagopyrum tataricum G.) revealed that two proteins reacted with the antiserum. The immunoblotting and genomic Southern analysis data indicated the rutinase gene were encoded by gene family. In an E.coli, and a S.pombe expression system, the translation product of a cDNA encoding the rutinase showed little flavonol 3-glucosidase activity.

Research Products

• [Journal Article] Molecular Characterization of a 10-kDa Buckwheat Molecule Reactive to Allergic Patients' IgE (2004)
  • Author(s): R.Matsumoto et al.
  • Journal Title: Allergy 59 Pages: 533-538
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Quantification of a major allergenic protein in common buckwheat cultivars by an enzyme-linked immunosorbent assay (ELISA) (2004)
  • Author(s): W.Maruyama-Funatsuki et al.
  • Journal Title: Fagopyrum 21 Pages: 39-44
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Molecular Characterization of a 10-kDa Buckwheat Molecule Reactive to Allergic Patients' IgE (2004)
  • Author(s): Matsumoto, R., Fujino, K., Nagata, Y., Hashiguchi, S., Ito, Y., Aihara, Y., Takahashi, Y., Maeda, K, Sugimura, K.
  • Journal Title: Allergy 59 Pages: 533-538
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Quantification of a major allergenic protein in common buckwheat cultivars by an enzyme-linked immunosorbent assay (ELISA) (2004)
  • Author(s): Maruyama-Funatsuki, W., Fujino, K., Suzuki, T, Funatsuki, H.
  • Journal Title: Fagopyrum 21 Pages: 39-44
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] R.Matsumoto et al.: "Molecular Characterization of a 10-kDa Buckwheat Molecule Reactive to Allergic Patients' IgE"Allergy. (In press).